The neuromuscular junction (NMJ), formed between motoneurons and skeletal muscle fibers, is one of the most studied synaptic structures (Witzemann 2006). In a mammalian vertebrate, whenever an action potential is fired by a motoneuron, pre-synaptic vesicles loaded with the neurotransmitter acetylcholine (ACh) are released in the synaptic cleft (Chow et al., 1985). The released ACh diffuses across the synaptic cleft and binds to the post-synaptic terminals in the muscle enriched with receptors for acetylcholine (AChRs). This leads to muscle contraction. In this transmission process the electrical impulses (action potentials) generated by the motoneuron are converted to chemical signals, then the chemical signals are converted into a mechanical signal in the form of muscle contraction. Therefore, not only do NMJs represent an important system for studying synapse formation and maturation, but also for studying how cells interconvert messages between electrical, chemical and mechanical modalities.
In vivo, NMJ formation is a multistep process, requiring the spatial and temporal interaction of growth factors, hormones and cellular structures that results in a pre-synaptic axonal terminal interfaced with a region of the skeletal muscle membrane (postsynaptic) pre-patterned with AChRs (Colomar et al., 2004; English 2003). In vitro culture models represent a powerful cell biology tool to study the role of these different growth factors, hormones and cellular structures involved in NMJ formation in a defined, controlled system. Consequently, the development of an in vitro system resulting in NMJ formation would facilitate investigations into the roles of specific factors involved in, and required for, the process to occur efficiently.
However, limited success has been achieved in developing a long-term in vitro system for NMJ formation in the absence of serum containing media and biological substrates. These issues limit the reproducibility of in vitro studies and their translation to tissue engineering applications and high-throughput assay development. For example, the concentration and/or temporal application of medium components could be investigated to determine their influence on NMJ formation, maturation and maintenance. Such a system also benefits from the absence of factors that may be present in serum that would inhibit these processes. Employing a non-biological growth substrate such as trimethoxysilylpropyl diethylenetriamine (DETA) provides an additional measure of control. DETA is a silane molecule that forms a covalently bonded monolayer on glass coverslips, resulting in a uniform, hydrophilic surface for cell growth. The use of DETA surfaces is advantageous from a tissue engineering perspective because it can be covalently linked to virtually any hydroxylated surface, it is amenable to patterning using standard photolithography (Ravenscroft et al., 1998) and it promotes long-term cell survival because it is non-digestible by matrix metalloproteinases secreted by the cells (Das et al., 2004; Das et al., 2007 (Nat. Protocols)). It is also possible that its structural relationship to the growth factor spermidine, which has recently been shown to prolong cell life (Eisenberg et al., 2009), contribute to its unique ability to enable long-term healthy cell cultures.
Previously, we developed a defined in vitro model facilitating the short-term co-culture of motoneurons and skeletal muscle that resulted in NMJ formation (Das et al., 2007 (Neuroscience)). This model also utilized a biocompatible silane substrate and a serum-free medium formulation. However, further improvements were necessary to enhance the physiological relevance of the NMJ development system. Limitations of the previous model were that it did not support long-term tissue engineering studies and therefore, could not mimic several of the muscle maturation processes observed in vivo by obtaining myotubes that more accurately represent mature extrafusal fibers.
As noted, neuromuscular junction (NMJ) formation, occurring between motoneurons and skeletal muscle, is a complex multistep process involving a variety of signaling molecules and pathways. In vitro motoneuron-muscle co-cultures are powerful tools to study the role of different growth factors, hormones and cellular structures involved in NMJ formation. In this study we have demonstrated a co-culture system that enable sarcomere assembly in the skeletal muscle myotubes as evidenced by A band/I band formation, increased NMJ density and selective myosin heavy chain (MHC) class switching. These results suggest we have discovered a group of biomolecules that act as molecular switches promoting NMJ formation and maturation as well as skeletal muscle fiber maturation to the extrafusal phenotype. This model system will be a powerful tool in basic NMJ research, tissue engineered NMJ systems, bio-hybrid device development for limb prosthesis and in regenerative medicine. It could also be useful in new screening modalities for drug development and toxicology investigations.